Method for characterizing a highly parallelized liquid handling technique using microplates and test kit for carrying out the method

ABSTRACT

A method for characterizing highly parallelized liquid handling technology using microplates and test kit for carrying out the method. Possibilities for characterizing a multi-channel or many-channel handling technology are known in principle. However, especially with regard to more highly parallelized devices and very small volumes, particularly under conditions corresponding to the actual application conditions, these possibilities were relatively expensive and presented problems especially with respect to the accuracy of evaluation and with respect to correctness and precision. The present method enables a more economical and very exact characterization for applications of this kind. According to the invention, a) a mean sample volume or mean reagent volume is determined by gravimetry from the totality of sample liquid or reagent liquid of all individual channels of the liquid handling technology; b) a normalized mean optical intensity is formed from optical measurement signals of all sample volumes or reagent volumes, each of which is mixed with a diluent; c) the volume accuracy of every individual channel of the liquid handling technology with respect to the mean sample volume or mean reagent volume is determined from the intensity deviation of the normalized optical measurement signal of the individual channel in relation to the normalized mean optical intensity. Further, a test kit is provided for advantageous implementation of the method. The invention is used wherever highly parallelized liquid handling technology is to be characterized with respect to accuracy and precision, particularly when the handled volumes lie within the μl range or sub-μl range and characterization is to be carried out under conditions approximating the real operating conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of International Application No.PCT-DE03/00834, filed Mar. 13, 2003 and German Application No. 102 12557.0, filed Mar. 14, 2002, the complete disclosures of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to a method for characterizing a highlyparallelized liquid handling technology using microplates and a test kitfor carrying out the method. It is used wherever highly parallelizedliquid handling technology is to be characterized with respect toaccuracy and precision, particularly when the handled volumes lie withinthe μl range or sub-μl range and the characterization must be carriedout under conditions corresponding to the real operating conditions ofthe liquid handling technology. Liquid handling technology using a largenumber of channels for sample handling, e.g., multipipettes with 96,384or more channels, provides a large number of sample volumes inindividual wells of a microplate that are arranged in grids forpreparation and evaluation of samples in many channels or storage andtransport, etc., thereof. The correctness and accuracy of samplehandling is critically important for the quality and usability of theanalysis results. For this reason, a check criterion which might behelpful as a measure of quality, as a basis for certification, and thelike, becomes increasingly important for the supplier and for the userof such liquid handling technology

b) Description of the Related Art

In recent years, highly parallelized, extremely miniaturized methods ofanalysis based on microplate technology have led to the development of alarge number of new and effective applications, particularly fortarget-oriented active ingredients for analysis of genomes and proteomesand for numerous other areas of biotechnology, medicine andenvironmental research. For the reasons described above, a correspondinghighly parallelized technique for many-channel dispensing, readertechnology and other technological developments capable of being adaptedto the latter which could be characterized in a manner approachingapplication as closely as possible were created for many-channelhandling of samples. Further, the characterization of a large number ofindividual channels should be practicable, must not require excessiveexpenditure on analysis and should be sufficiently precise.

As has been well known for a long time with respect to a large quantityof pipettes, gravimetric methods are used for this characterizationalong with methods which measure the dilution of an analytic signal of asample by a diluent, this analytic signal being, in itself, easy totrack. Examples of signals of this type are optical signals orradioactivity of a sample.

Gravimetric methods are very precise, but are hardly usable for the μlrange and sub-μl range insofar as assessments of precision and accuracyare required to fall within a range of better than 0.5%. Further, theuse of gravimetric methods is rendered nearly impossible firstly byevaporation, which constitutes a severe hindrance especially within thisvolume range, and secondly by problems relating to practicability (verymany individual channels must now be characterized, whereas previouslyonly 1 to a maximum of 12 channels had to be characterized).Examinations of this kind were previously restricted to relatively largeindividual volumes (e.g., GIT Laborzeitschrift 11/2001, 1185-86). Therealization of conditions approximating those of real application alsocreates problems for characterization.

Photometric methods for calibrating pipettes were already described inthe 1980s. For example, U.S. Pat. No. 4,354,376 describes a kit forcalibrating pipettes which is based on the principle of measuring thedilution of dye solutions. U.S. Pat. No. 5,492,673 describes a reagentsystem for colorimetric calibration of pipettes which uses a specialmixture of substances to correct for the path length of the round cellsor cuvettes that are used in order to prevent nonlinearities in themeasured absorbances as a function of dye concentration brought about byagglomeration and to improve the stability of the proposed reagent kit.The mixture comprises a 2-buffer system, each with a color indicator.These color indicators differ sharply in the position of the wavelengthsof the maxima of the light absorption. Further, the mixture containssubstances which inhibit aglomerization and improve stability. However,considerable expenditure on correction is required to excludedevice-specific influences due to the photometer that is used and due tothe cuvettes and influences particularly of the surrounding temperature.

The availability of parallel reader technology invites thecharacterization of parallelized dispensing technique using thistechnology, especially since the large number of individual channels tobe characterized would otherwise be very difficult to calibrate.However, it must be taken into consideration that reader technology formicroplates is based on the principle of vertical photometry; that is,it has no fixed path length for the individual well. The path length isdetermined by the volume that is used and by the developing meniscus andis subject to considerable variability depending upon the surfacecharacteristics of the analyte and the mechanical conditions under whichthe microplate is handled. In U.S. Pat. No. 6,188,476, the absorbance ofwater in the infrared range is made use of to normalize the measuredabsorbances of the analyte with respect to a uniform path length inorder to compensate for uncertainty regarding the path length. However,practical experience shows that although the average path length can bedetermined by this correction, compensation of the influence of theindividual menisci is unsatisfactory.

In the simplest case, when an absorbance-measuring reader is used tomeasure the sample volume of a channel of the liquid handling technologywith n channels, a diluent volume V_(D) is introduced in the individualwells of the microplate and a sample volume V_(P) which contains a dyeF₁ in a concentration C_(PF1) and which is to be determined is addedthereto and mixed. The measured n absorbances of the sample solutionA_(P) and the n mixtures A_(M) are functions of the respectiveconcentrations of solutions and the path length d according to theBeer-Lambert law:A _(P)=ε_(F1) *C _(PF1) *d _(P)A _(M)=ε_(F1) *C _(MF1) *d _(M),where C_(MF1) is the concentration of dye F₁ in the mixture and εdesignates the extinction coefficient of the indicator.

The dilution factor D_(F)D _(F) =C _(MF1) /C _(PF1) =V _(P)/(V _(P) +V _(D))can be used to determine V_(P).V _(P) =V _(D) *DF/(1−DF)=V _(D) *C _(MF1) /C _(PF1)/(1−C _(MF1) /C_(PF1))  (1)

It can be seen from equation (1) that the precision with which V_(P) isdetermined depends upon the precision with which two absorbances aredetermined in the reader and upon the accuracy of the present volumeV_(D).

The accuracy of the absorbances A measured in the reader depends uponmultiplicatively acting (f) and additively acting (a) errors:A=A*f+a.

Multiplicatively acting errors are chiefly the path length which variesbecause of meniscus formation and the temperature-dependent changes inε; additively acting errors are brought about, for example, by theformation of bubbles, which is frequently observed, and by deposits,scratches and fizz or lint which are sometimes observed. These errors,which ultimately influence the analytic results of the volumedetermination, can be eliminated in large part by multiwavelengthphotometry. A procedure of this kind is described, for example, fordetermining temperature in microplates with thermochrome indicators byabsorbance measurement (DE 199 28 056). Practical investigations of thevariability of the absorbances measured in readers show that while goodprecision of the relative values of the absorbances in the individualwells of a microplate with respect to the mean of all measured wells(intra-assay precision) is achieved through the use of multiwavelengthphotometry, the individual values and mean values of the absorbancesmeasured and obtained, respectively, for different plates haveunacceptably high deviations.

Further, with regard to the use of reader technology for characterizingmultipipettes, it must be taken into consideration that there arepresently no readers for microplates with well densities greater than384 per microplate (particularly 1536 or more wells) which can measurelight absorbances with sufficient accuracy.

OBJECT AND SUMMARY OF THE INVENTION

Therefore, it is the primary object of the invention to provide a methodby which highly parallelized liquid handling technology, preferably forvolumes in the μl range and sub-μl range, can be characterized moreeconomically and with greater exactness with respect to accuracy andprecision under conditions which come very close to the prescribed useof this liquid handling technology.

Further, a test kit is to be provided for the practicable implementationof the method.

This object is met in that sample volumes are dispensed in the wells ofat least one microplate with the liquid handling technology to becharacterized, wherein conditions coming as close as possible to aprescribed use of the liquid handling technology to be characterized andof the microplate are selected with respect to the selection,constitution and amount of sample liquid or reagent liquid (pipettingsolution) and diluent and with respect to the dispensing, handling andanalysis of the samples.

According to the invention, a mean sample volume or mean reagent volumewith respect to the number of channels of the liquid handling technologyis determined by gravimetry under conditions of greatly reducedevaporation from all of the sample volumes and reagent volumes of theindividual wells that are dispensed simultaneously in the wells of themicroplate; every sample volume and reagent volume, each containing forthe purpose of optical measurements a first indicator with specificoptical characteristics and at least a second indicator with opticalcharacteristics differing from those of the first indicator, is mixedwith a diluent which contains the same at least second indicator in aconcentration identical to that of the sample volume or reagent volumebut which does not contain the first indicator; at least one firstoptical measurement signal caused by the first indicator is generatedand a second optical measurement signal caused by the second indicatoris generated in the same way for each well of the microplate that isfilled with a mixture of the sample volume or reagent volume anddiluent; every first optical measurement signal is scaled or normalizedwith respect to its intensity by means of the at least second opticalmeasurement signal of the associated well of the microplate for purposesof error compensation, particularly for eliminating influences of thewell geometry, the shape of the liquid surface in the well and blankabsorbances due to scratches, bubbles and lint, etc.; a mean opticalintensity value which is normalized with respect to the quantity ofchannels of the liquid handling technology to be characterized is formedfrom all normalized optical measurement signals and the intensitydeviation of the corresponding normalized optical measurement signal inrelation to the normalized mean optical intensity value is acquired forevery well of the microplate; and the volume of every channel of theliquid handling technology to be characterized is determined withreference to the mean sample volume or mean reagent volume determined bygravimetry from the intensity deviation of the corresponding normalizedoptical measurement signal in relation to the mean optical intensityvalue.

With the proposed method, a mean value of all sample volumes or reagentvolumes dispensed in the microplate simultaneously by the liquidhandling technology to be characterized is first determined. This meansample volume or mean reagent volume is preferably determined by highlyaccurate, repeated weighing, wherein the dispensing of the samplevolumes or reagent volumes by the liquid handling technology can becarried out in empty wells of the microplate and in a microplate whosewells already contain diluent.

Said optical measurement signals and the relative evaluation of thechannel-specific intensity deviations from a normalized mean value ofoptical intensity can also be determined with high accuracy for preciseevaluation. The intensities of at least two individual opticalmeasurement signals are measured at different wavelengths for every wellof the microplate, the at least second optical measurement signal beingused to eliminate the above-mentioned well-oriented interference factorswhen normalizing the first optical measurement signal. Buffers whose pHvalues change very slightly or not at all with changes in temperatureare preferably used to reduce the possible influence of differenttemperatures in the individual wells of the microplate due to thetemperature dependence of the extinction coefficient ε of theindicators.

With respect to the very exactly determinable mean sample volume andmean reagent volume of all of the sample volumes and reagent volumesdispensed in the microplate by liquid handling technology, the volumeaccuracy of every channel of the liquid handling technology can bedetermined with great precision from the intensity deviation of thenormalized well-specific optical measurement signal in relation to thenormalized mean optical intensity value. The mean sample volume and meanreagent volume are proportional to the mean intensity value of thenormalized optical measurement signal, so that the volume deviation and,therefore, also the sample volume determined for every well forcharacterizing the liquid handling technology can be calculated from therespective channel-oriented intensity deviation of the correspondingnormalized optical measurement signal in relation to the mean intensityvalue of all normalized optical measurement signals.

For purposes of a clear visual characterization of the liquid handlingtechnology, said measurement signal intensity deviations can bepresented as so-called false-color presentations in a matrixcorresponding to the channel geometry of the liquid handling technology.

Representing the above-mentioned well-specific sample volumes andreagent volumes determined for characterizing the liquid handlingtechnology in a matrix of this kind which corresponds to the channelgeometry of the liquid handling technology is also advisable forevaluation.

It is advantageous that the characterization of the liquid handlingtechnology can be carried out under conditions which come very close toa prescribed use of the liquid handling technology and of the microplatewith respect to the handling and analysis of samples. These conditionsare realized in particular through suitable selection, constitution andvolumes of sample liquid and diluent and through adequate dispensing,handling and analysis of samples in themselves. Therefore, the resultsdetermined with the method according to the invention are highlyrelevant not only for producers of liquid handling equipment (thefoundations for appropriate standards would be established in this way),but also, owing to the fact that it approximates practical conditions,for users of this liquid handling technology and accordingly also forinterpreting analysis results.

Further embodiments of the method according to the invention areindicated in dependent claims 2 to 29.

Further, a special test kit is indicated for advantageously carrying outthe method.

The kit comprises handling instructions and at least five preparedsolutions (L_(1a), L_(2a), L_(1f), L_(2f), L₃) from which the samplevolumes and reagent volumes and the diluent can be produced in a mannerspecific to the application. Two solutions (L_(1a), L_(2a),) areprovided for photometric measurements and two solutions (L_(1f),L_(2f),) are provided for fluorescence measurements. Solutions L_(1a),L_(1f), are parent solutions for the respective first optical indicatorand solutions L_(2a), L_(2f), are parent solutions for the respectivesecond optical indicator. Solution L₃ is a parent solution for aquasi-temperature-insensitive buffer (0.5 to 1 M phosphate buffer, pH11.0).

All kit solutions are prepared in highly concentrated form in comparisonto the working concentration.

For photometry, solution L_(1a) comprises 30 mM to 300 mM p-nitrophenolin 96% (vol/vol) ethanol and solution L_(2a), comprises 30 mM to 300 mMphenolphthalein in 96% (vol/vol) ethanol.

For fluorimetry, solution L_(1f) comprises 30 mM to 300 mMmethylumbelliferone in dimethyl sulfoxide and solution L_(2f) comprises0.3 mM to 30 mM fluorescein in 0.1 M phosphate buffer pH 11.0.

In the following, the invention will be described more fully withreference to application examples shown in the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 shows the temperature dependence of the absorbance ofp-nitrophenol in diethanolamine buffer (Application Example 4);

FIG. 2 shows the temperature dependence of the absorbance ofphenolphthalein in diethanolamine buffer (Application Example 4);

FIG. 3 shows the temperature dependence of the absorbance ofp-nitrophenol in phosphate buffer (Application Example 4);

FIG. 4 shows the temperature dependence of the absorbance ofphenolphthalein in phosphate buffer (Application Example 4);

FIG. 5 shows standard deviations of different individual masses from 15individual weighings in microplates (Application Example 9);

FIG. 6 shows a representation of the quotient A1/A2 for every well ofthe microplate in the form of a matrix (Application Example 10); and

FIG. 7 shows a representation of the sample volumes and reagent volumes(μl) calculated for every well of the microplate from the mean samplevolume and mean reagent volume determined by gravimetry and the relativephotometric deviations in the form of a matrix (Application Example 10).

DESCRIPTION OF THE PREFERRED EMBODIMENTS AND EXAMPLES ApplicationExample 1

A 384-channel dosing device serving as an example for a liquid handlingtechnology is to be characterized under conditions approximatingapplication for its prescribed use by means of absorbance measurements.

First, 384×48 μl of a diluent (solution D, 0.1 M phosphate buffer, pH11.0 with 0.04 mM phenolphthalein) which was produced from solutionsL_(2a) and L₃ of a kit in accordance with the handling directions arepipetted into the wells of two 384-well microplates by a precisiondosing device, these microplates being covered by a tightly closingcover. The first microplate is used for evaluation of the dosing; thesecond microplate is used for determining the evaporation loss withinthe handling period. The two microplates are then weighed in order todetermine a dead weight or empty weight m₁ of the first microplate andan initial weight m_(a) of the second microplate.

After weighing, 2 μl of a sample solution P are pipetted into the wellsalready containing solution D of the first microplate by the 384-channeldosing device to be characterized. Solution P comprises 0.1 M phosphatebuffer, pH 11.0, with 0.04 mM phenolphthalein and 0.06 mM p-nitrophenoland was produced from solutions L_(1a), L_(2a) and L₃ of theabove-mentioned kit according to handling directions.

All of the handling processes for the above-mentioned pipetting ofsolution P are also carried out with the second microplate forreference, but without actually pipetting the solution P into the wellsalready containing solution D of the second microplate. Next, the twomicroplates are weighed again with their covers. The new weight of thefirst microplate m₂ corresponds to the sum of the empty weight (m₁) andthe added weight (Δm) caused by the pipetting process minus theevaporation (m_(υ)); the new weight (final weight) of the secondmicroplate m_(e) results from the initial weight m_(a) minus the weightloss due to the evaporation weight (m_(υ)). This gives the followingequations:Δm=m ₂ −m ₁ +m _(υ) and m _(υ) =m _(a) −m _(e).

Further, the density ξ of the pipetting solution (solution P) isdetermined by weighing with a pycnometer, known per se, and the meanvolume V_(P) of solution P dispensed by pipetting into the wells of thefirst microplate is calculated with respect to all channels of the384-well dosing device.V _(P) =Δm/(ξ*384)=(m ₂ −m ₁ +m _(a) −m _(e))/(ξ*384)

The first microplate is agitated in an agitator, known per se, forapproximately 60 minutes so that the solutions D and P located in thewells are thoroughly mixed and the absorbances of p-nitrophenol,phenolphthalein and the blank absorbance are subsequently determined byphotometric measurements at wavelengths of 405 nm, 540 nm and 620 nm.Quotient A₄₀₅/A₅₄₀ and differential quotient (A₄₀₅−A₆₂₀)/(A₅₄₀−A₆₂₀) areformed from the absorbance values measured for each well of the firstmicroplate at the above-mentioned wavelengths for normalizing theabsorbance of the first indicator p-nitrophenol with well-specific addedsignals for each well. A spreadsheet, known per se, is preferablyapplied for calculating and further processing these values.

The associated optical mean values are formed from all 384 quotients anddifferential quotients of the first microplate and the deviation of thequotient value or differential quotient value from the respectiveoptical mean value is acquired for every well. The relative deviationsof the channel-specific values from the mean values of the formedquotients and differential quotients are determined in this way. Thesedeviations are proportional to the relative channel-specific deviationsof the pipetted channel-specific sample volumes from the mean samplevolume of the first microplate determined by gravimetry.

Based on this proportionality, the ratio f=Ik/Im of the photometricintensity deviation of a well (Ik) from the photometric mean intensity(Im) can be related to the ratio f=Vk/Vm of the volume deviation (Vk) ofthe same channel from the mean volume (Vm). The pipetted volume of everychannel then corresponds to Vk=f*Vm.

False-color presentations of said relative deviation from the respectivemean value are helpful as a visual evaluation aid for fast, clearassessment.

Application Example 2

Intra-assay precision of the photometric measurement in 384-wellmicroplates for different dye concentrations:

Solutions of different p-nitrophenol concentrations are produced in thata p-nitrophenol parent solution in 0.1 M phosphate buffer (pH 11.0) with0.04 mM phenolphthalein is gradually diluted by 0.1 M phosphate buffer(pH 11.0) with 0.04 mM phenolphthalein. 50 μl of the p-nitrophenolworking solutions in various concentrations are introducedsimultaneously into eight different wells of a 384-well microplate by amultichannel dosing device and measured in a reader (SpektraFluor Plus,Tecan) at wavelengths of 405 nm (A1), 540 nm (A2) and 620 nm (A3), wherethe bandwidth of the filters is ±10.

The intra-assay precision of the respective 8 identical solutions islisted in the following Table 1. The absorbance at 540 nm (A2) lieswithin the range of 0.44 to 0.46.

It will be seen that the precision is highly dependent upon the absoluteabsorbance with a VK minimum in the absorbance range of 0.35 to 0.53.The precision is appreciably improved in this range by formingquotients. A further improvement in precision can be achieved by formingdifferential quotients only at high absorbances. The precisions actuallyfound in the optimal absorbance range indicate the maximum assertionthat can be achieved by the method with respect to the precision of theindividual channels with the indicated color system and the indicatedreader.

TABLE 1 Parameter A1 Mean 0.064 0.266 0.349 0.442 0.534 0.719 0.817 VK(%) 1.66 2.72 0.45 1.01 0.93 2.87 3.23 A1/A2 VK (%) 2.25 0.47 0.30 0.340.39 0.14 0.88 (A1-A3)/(A2-A3) VK (%) 5.14 0.79 0.31 0.39 0.41 0.19 0.16

Application Example 3

Inter-assay precision of the photometric measurement in 96-wellmicroplates for different dye concentrations:

Solutions of different p-nitrophenol concentrations are produced in thata p-nitrophenol parent solution in 0.1 M phosphate buffer (pH 11.0) with0.04 mM phenolphthalein is gradually diluted by 0.1 M phosphate buffer(pH 11.0) with 0.04 mM phenolphthalein. 150 μl of the p-nitrophenol worksolutions in various concentrations are introduced simultaneously inindividual wells of a 96-well microplate on three different days with amultichannel dosing device and measured in the reader at wavelengths of405 nm (A1), 540 nm (A2) and 620 nm (A3), where the bandwidth of thefilters is ±10.

The inter-assay precision of the three assays is shown in the next Table2. The absorbance at 540 nm (A2) lies within the range of 0.398 to 0.42.

The inter-assay precision shows a clear dependence on the absorbancelevel with a minimal VK around A=0.5. In the absorbance range from 0.4to 0.52, there is an appreciable improvement in the measuring precisionby quotient formation. However, the results also show that an exactdetermination of the accuracy (with a deviation ≦0.5%) for multichanneldosing devices in the entire volume range cannot be achieved solely byoptical means.

TABLE 2 Inter-assay variation coefficient (%) Absorbance (A1) A1 A1/A2(A1-A3)/(A2-A3) 0.048 3.05 3.21 5.13 0.43 0.82 0.70 0.82 0.52 0.59 0.330.40 0.61 0.22 0.59 0.79 0.68 2.67 2.85 3.06

Application Example 4

Temperature dependence of the photometric measurement signals ofp-nitrophenol (F1) and phenolphthalein (F2) in different buffer systems:

Both dyes were dissolved in 0.1 M diethanolamine buffer (pH 10) and alsoin 0.1 M phosphate buffer (pH 11.0) in such a way that an absorbance ofabout 0.5 is achieved with a path length of 1 cm. These solutions weremeasured repeatedly at different temperatures in a temperature-regulatedphotometer (Contron). The temperature was adjusted up and down by acirculating water bath and measured by a thermosensor in the measurementcuvette.

Diethanolamine buffer: The results are shown in FIG. 1 and FIG. 2. FIG.1 shows the temperature dependence of p-nitrophenol absorbance and FIG.2 shows that of the phenolphthalein absorbance. It was found that therewas a slight temperature-dependent change in the p-nitrophenolabsorbance (A1) compared to phenolphthalein. The phenolphthaleinabsorbance (A2) shows a clear linear drop of an average of 0.0073/K inthe range of 28° C. to 37° C. A linear increase in the absorbancequotient A1/A2 of 0.025/k is derived from this.

Phosphate buffer: It was found that in comparison to the diethanolaminebuffer there is a slight temperature-dependent linear increase in thep-nitrophenol absorbance of only 0.0003/K (FIG. 3) and a linear decreasein the phenolphthalein absorbance of 0.0001/K (FIG. 4) within the rangeof 25° C. to 39° C. This is less than one seventieth of the decrease inabsorbance of phenolphthalein in diethanolamine buffer. A very slightlinear increase in the absorbance quotient of 0.0008/K is derived fromthis. With a quotient of, e.g., 0.6, the latter value corresponds to adeviation of 0.13%/K and is therefore less than the measurementprecision found in known readers.

Application Example 5

Intra-assay precision of the photometric measurement in all wells of384-well microplates in the absorbance range of the highest precisionusing A1, quotient A1/A2 and differential quotient (A1−A3)/(A2−A3):

50 μl of a homogeneous mixture comprising 0.06 mM p-nitrophenol and 0.09mM phenolphthalein were pipetted into the wells of a 384-well microplatewith a multichannel dosing device, the plate was briefly agitated andthen measured in the reader at 405 nm (A1), 540 nm (A2) and 620 nm (A3).

The mean values, relative deviations, expressed as variationcoefficients, were calculated for the absorbance value A1 of all wellsand for their normalized values from A1/A2 and (A1−A3)/(A2−A3) anddetermined for each individual well. The results are compiled in Table3. It will be seen that the precision is appreciably improved throughnormalization by means of quotients. The forming of differentialquotients does not result in a further increase in precision in thiscase.

TABLE 3 Parameter Microplate A1 A2 A1/A2 (A1-A3)/(A2-A3) Mean 1 0.7050.560 1.26 1.26 SD 0.075 0.054 0.00 0.00 VK 1.06 0.96 0.26 0.26 Mean 20.700 0.553 1.27 1.28 SD 0.068 0.054 0.00 0.01 VK 0.97 0.98 0.33 0.39

Application Example 6

Intra-assay precision of the fluorimetric measurement using Flu1 andquotient Flu1/Flu2:

50 μl of a homogeneous mixture comprising 0.06 mM methylumbelliferoneand 1.3 μM fluorescein were pipetted into the wells of a 1536-wellmicroplate by a multichannel dosing device, the plate was brieflyagitated and then measured in a reader at 460 nm (Flu1, excitation 365nm) and 535 nm (Flu2, excitation 485 nm).

The mean values, standard deviations and variation coefficients werecalculated for the fluorescence values Flu1 and Flu2 of all wells andfor their normalized values from Flu1/Flu2. The results are compiled inTable 4. The considerable improvement in precision through the formationof quotients can be seen in this case also. The precision of thequotients shows the maximum precision that can be achieved by therespective reader and, therefore, the resolution of the method.

TABLE 4 Parameter Microplate Flu1 Flu2 Flu1/Flu2 Mean 1 5370.51 8142.270.660 SD 384.41 578.92 0.008 VK 7.16 7.11 1.19 Mean 2 5410.47 8331.780.650 SD 551.92 866.15 0.008 VK 10.20 10.40 1.20

Application Example 7

Intra-assay precision of pipetting of 384 sample volumes usingmeasurement signals A1 and quotient A1/A2.

Different volumes, indicated in column 2 of the following Table 5, ofthe sample solutions P comprising p-nitrophenol in the concentrationsindicated in column 1 of Table 5 and, in addition, 0.04 mMphenolphthalein were dispensed in the wells of 384-well microplates by amultichannel dosing device to be checked. For this purpose, volumes of asolution D of 0.04 mM phenolphthalein in 0.1 M phosphate buffer (pH11.0) were distributed in the individual wells by another precisionmultichannel dosing device in such a way that the total volume in everywell was 50 μl. The microplates were tightly closed with adhesive foilsand agitated for 60 minutes. The absorbances were then measured in thereader at wavelengths of 405 nm (A1) and 540 nm (A2).

The mean values, standard deviations and variation coefficients (VK)were calculated for the absorbance values A1 and A2 of all wells and fortheir normalized values from A1/A2. The results are compiled in Table 5.

The precisions normalized by forming quotients are better in all casesthan the precisions calculated by exclusive use of A1 and thereforerepresent the dosing accuracy better than the measurement of A1exclusively.

TABLE 5 Solution P p-Nitrophenol Reference concentration volume Quotient(mM) (μl) Parameter A1 A2 A1/A2 0.3 10 Mean 0.566 0.455 1.245 SD 0.0070.004 0.008 VK 1.194 0.906 0.667 0.3 10 Mean 0.565 0.494 1.144 SD 0.0050.003 0.008 VK 0.823 0.702 0.719 0.6 5 Mean 0.568 0.498 1.14 SD 0.0080.006 0.015 VK 1.348 1.237 1.324 0.6 5 Mean 0.549 0.49 1.117 SD 0.0060.005 0.012 VK 1.183 1.029 1.045

Application Example 8

Intra-assay precision of pipetting of 384 sample volumes using Flu1 andquotient Flu1/Flu2:

Sample solutions comprising methylumbelliferone and 2 μM fluorescein in0.1 M diethanolamine buffer (pH 9.8) were dispensed in the wells of384-well microplates by a multichannel dosing device to becharacterized. The methylumbelliferone concentration is variable and isgiven in column 1 of Table 6; the volume is given in column 2. Inaddition, different volumes of a solution of 2 μM fluorescein in 0.1 Mdiethanolamine buffer (pH 9.8) were pipetted in the wells of themicroplates by another precise multichannel dosing device. These volumeswere selected in such a way that a final volume of 50 μl resulted. Themicroplates were agitated for 60 minutes and then measured in a readerat wavelengths of 460 nm (Flu1, excitation 365 nm) and 535 nm (Flu2,excitation 485 nm).

The mean values, standard deviations and variation coefficients werecalculated for the fluorescence value Flu1 of all wells and for theirnormalized value from Flu1/Flu2. The results are compiled in Table 6. Itwill be noted that the precision is mostly improved after formingquotients.

TABLE 6 Methyl- umbelliferone Reference concentration volume Para-Quotient (Mm) (μl) meter Flu1 Flu2 Flu1/Flu2 0.6 5 Mean 18447.6 250000.738 SD 391.7 503.9 0.014 VK 2.12 2.01 1.89 0.6 5 Mean 18266 253760.720 SD 368.4 498.9 0.012 VK 2.02 1.966 1.67 3 1 Mean 17581 24983 0.704SD 608.7 604.8 0.025 VK 3.46 2.42 3.58 3 1 Mean 18131 25096 0.723 SD586.5 593.4 0.023 VK 3.24 2.36 3.17

Application Example 9

Precision of weighing: Various dry individual masses were determined 15times by a precision scale using microplates with 384 wells and 1536wells as carriers. The standard deviations from 15 individual weighingsfor different individual masses are compiled in FIG. 5. For weighingwith the microplate empty weight, for 384-well microplates of about 56 gand for 1536-well microplates of about 34 g, there is a mean standarddeviation in the weighing for individual masses within the range of5-1300 mg of 0.0699 mg and 0.0618 mg (FIG. 5).

When weighing in microplates, the standard deviation is relativelyconstant and not dependent upon the individual mass (FIG. 5).

This means that there are different relative errors, albeit very slight(compare Table 7), for different pipetted individual volumes. In theextreme case (384 times 0.05 μl sample volume in 384-well microplates),there is a mean error of less than 0.4%; for all other volumes,appreciably smaller errors in weighing accuracy are to be expected.Therefore, the weighing error due to scale inaccuracies is negligiblewithin the volume ranges and mass ranges being examined. Therefore,weighing is the method of choice for determining dosing accuracy.

TABLE 7 Computational error in weighing differential weights inmicroplates (MP) Individual Total dosing volume Computational error dueto dosing per plate weighing inaccuracy volumes (μl ≅ mg) (% of totaldosed volume) (μl) 384-well MP 1536-well MP 384-well MP 1536-well MP0.05 19.2 76.8 0.3642 0.0805 0.1 38.4 153.6 0.1821 0.0403 0.2 76.8 307.20.0911 0.0201 0.5 192 768 0.0364 0.0081 0.7 268.8 1075.2 0.0260 0.0058 1384 1536 0.0182 0.0040 2 768 3072 0.0091 0.0020

Application Example 10

Evaluation of the accuracy and precision of a 384-channel dosing device:

The method principle applied in Application Example 1 is used. Twopipetter variants a and b approximating application are evaluated inparallel with 1 μl being dosed by way of example.

Pipetter variant a: Solution P is pipetted into a dry microplate. 49 μlof solution D are dispensed subsequently in all wells.

Pipetter variant b: Solution P is pipetted into a microplate filled with49 μl of solution D.

Solution P comprises 0.04 mM phenolphthalein and 3 mM p-nitrophenol indimethyl sulfoxide; solution D comprises 0.04 mM phenolphthalein and 0.1M phosphate buffer (pH 11.0).

The procedure and the results of the gradual steps are shown in Table 8and in FIG. 6 and FIG. 7.

TABLE 8 Variant a Variant b Variant a Variant b Step 1 Assertion 49 μlof a solution D are pipetted into the wells of two 384- well MPs whichare covered by a tightly closing cover The empty MP with MP1 and MP2 arem₁: 69.0718 g m₁ (MP1): 87.9158 g cover is weighed weighed with coversm_(a) (MP2): 87.8633 g 1 μl of solution P is 1 μl of solution P ispipetted into the dry pipetted into the wells of the MPs filled wells ofMP1 with the dosing device to be tested; parallel identical handling ofMP2, but without pipetting The MPs are weighed Both Mps are m₂: 69.4895g m₂(MP1): 88.3232 g again with covers weighed again with Δm = m₂ − m₁ =M_(e)(MP2): 87.8516 g covers, cover of MP 417.7 mg Δm = m₂ − m₁ + m_(υ)= 1 is then replaced 419.1 mg with adhesive foil m_(υ): 11.7 mg 49 μl ofsolution D are pipetted into the wells, covered with adhesive foilNear-time determination of density ξ of Differential weight for 5.0 mlpycnometer pipetting solution contents: 5.4285 g Empty weight ofpycnometer: 38.5644 g Density ξ = 1.0857 g/ml Weight of pycnometerfilled with solution P: 43.9929 g Calculation of mean actual volume(69.4895 g − 69.0718 g)/ (88.3232 g − 87.9158 g + (1.0857 g/ml * 384) =87.8633 g − 87.8516 g)/ 1.0019 μl (1.0857 g/ml * 384) = 1.0053 μlMixture MP Mixture MP1 Step 2 MP is measured in a MP1 is measured in areader at 405 nm and reader at 405 nm and 540 nm 540 nm Quotient(A₄₀₅/A₅₄₀) formed for the associated absorbances of every well Step 3Mean MP value taken from all 384 quotients Mean value: 1.234 Mean value:1.342 (photometric mean intensity) and the relative SD: 0.041 SD: 0.028deviation of every well value from the VK: 3.35 VK: 2.07 photometricplate averages is calculated

FIGS. 6 and 7 show a false-color presentation of the relative deviationfrom the photometric plate average and the actual dosing volume derivedtherefrom for every well of the microplate 1 and, therefore, for everyassociated channel of the dosing device.

FIG. 6 represents the quotient A1/A2 for every well and FIG. 7represents the dosing volume calculated from the mean sample volumedetermined by gravimetry and the channel-specific relative photometricdeviations.

In FIGS. 6 and 7, a medium box color represents a well whose valuedeviation lies within the range of the plate average ±1 of the standarddeviation; a light box color represents a well whose value deviationdeviates downward by more than 1 SD from the plate average; a dark boxcolor represents well values showing values that deviate upward by morethan 1 SD from the plate average.

While the foregoing description and drawings represent the presentinvention, it will be obvious to those skilled in the art that variouschanges may be made therein without departing from the true spirit andscope of the invention.

1. A method for characterizing highly parallelized liquid handlingtechnology, preferably for volumes in the μl range and sub-μl range,using microplates, wherein the liquid handling technology has a largenumber of elements for sample handling, such as multipipettes, by whicha large number of sample volumes of a sample liquid or reagent liquidare placed in wells of a microplate that are arranged in grids forpreparation and analysis or storage and transport, etc., of samples inmany channels with determination of volumes by gravimetry and opticalmeasurement in the presence of a diluent, comprising the steps of:determining a mean sample volume or mean reagent volume with respect tothe number of channels of the liquid handling technology by gravimetryunder conditions of greatly reduced evaporation from all of the samplevolumes or reagent volumes that are dispensed simultaneously into thewells of the microplate by the liquid handling technology to becharacterized; mixing every sample volume and reagent volume, eachcontaining a first indicator with specific optical characteristics andat least one second indicator with optical characteristics differingfrom those of the first indicator for purposes of optical measurements,with a diluent which contains the same at least second indicator in aconcentration identical to that of the sample volume or reagent volumebut which does not contain the first indicator; generating at least onefirst optical measurement signal caused by the first indicator and, inthe same way, a second optical measurement signal caused by the secondindicator for each well of the microplate that is filled with a mixtureof the sample volume or reagent volume and diluent; normalizing everyfirst optical measurement signal with respect to its intensity by the atleast second optical measurement signal of the associated well of themicroplate for purposes of error compensation, particularly foreliminating influences of the well geometry, the shape of the liquidsurface in the well, the blank absorbances, etc.; forming a mean opticalintensity value which is normalized with respect to the quantity ofchannels of the liquid handling technology to be characterized from allnormalized optical measurement signals and the intensity deviation ofthe corresponding normalized optical measurement signal from thenormalized mean optical intensity value is acquired for every well ofthe microplate; and determining the volume of every channel of theliquid handling technology to be characterized with reference to themean sample volume or mean reagent volume determined by gravimetry fromthe intensity deviation of the corresponding normalized opticalmeasurement signal in relation to the mean optical intensity value. 2.The method according to claim 1, wherein the mean sample volume or meanreagent volume is determined by repeated weighing, wherein the densityof the sample volumes or reagent volumes is known.
 3. The methodaccording to claim 2, wherein the weighing is carried out before mixingthe sample volumes or reagent volumes with the diluent.
 4. The methodaccording to claim 2, wherein the weighing is carried out after mixingthe sample volumes or reagent volumes with the diluent.
 5. The methodaccording to claim 1, wherein the dispensing of the sample volumes orreagent volumes by the liquid handling technology to be characterizedinto the wells of the microplate is carried out in a steam-saturatedchamber in order to create conditions of greatly reduced evaporation. 6.The method according to claim 1, wherein the microplate is closed, forexample, by a cover or by a foil, so as to be protected againstevaporation after the sample volumes are dispensed into its wells. 7.The method according to claim 1, wherein the dispensing of the samplevolumes or reagent volumes by the liquid handling technology to becharacterized is carried out in the wells of a closed microplate whoseclosure can be perforated.
 8. The method according to claim 1, wherein aloss of mass occurring as a result of evaporation is continuouslydetermined by weighing, and wherein the loss of mass and the course ofevaporation over time is deduced by extrapolation from the initialvolume, preferably using computation technology.
 9. The method accordingto claim 1, wherein the dispensing of the sample volumes or reagentvolumes by the liquid handling technology to be characterized is carriedout in empty wells of the microplate, and wherein the diluent volumesare pipetted into the latter subsequently.
 10. The method according toclaim 1, wherein the dispensing of the sample volumes or reagent volumesby the liquid handling technology to be characterized is carried out inwells of the microplate in which the diluent volumes have already beenintroduced.
 11. The method according to claim 1, wherein an aqueoussolution is used as sample liquid or reagent liquid.
 12. The methodaccording to claim 1, wherein a solution of organic solvent is used assample liquid or reagent liquid.
 13. The method according to claim 11,wherein a mixture of organic solvents and aqueous solutions is used assample liquid or reagent liquid.
 14. The method according to claim 1,wherein an aqueous solution is used as diluent.
 15. The method accordingto claim 1, wherein a solution of organic solvent is used as diluent.16. The method according to claim 1, wherein a mixture of organicsolvents and aqueous solutions is used as diluent.
 17. The methodaccording to claim 1, wherein additional reagents such as buffers,saline solutions, protein-containing solutions, cell suspensions,particle solutions and different solvent proportions are added to thesample liquid or reagent liquid and/or to the diluent.
 18. The methodaccording to claim 17, wherein a phosphate buffer with a pH of 9.0-12.5which is virtually independent from temperature with respect to its pHvalue is added as a buffer.
 19. The method according to claim 1, whereindyes are used as indicators in the sample liquid or reagent liquid or inthe diluent.
 20. The method according to claim 19, wherein p-nitrophenoland phenolphthalein are used as dyes.
 21. The method according to claim20, wherein p-nitrophenol, as first indicator, is added only to thesample liquid or reagent liquid, and wherein phenolphthalein, as secondindicator, is added in an identical concentration to the sample liquidor reagent liquid and to the diluent.
 22. The method according to claim19, wherein the normalized optical measurement signal is generated basedon the formation of the quotient from the first optical measurementsignal absorbance A1 and the second optical measurement signalabsorbance A2 (A1/A2) in the wavelength range of 390-420 nm (A1) and inthe wavelength range of 530 to 560 nm (A2).
 23. The method according toclaim 19, wherein the normalized optical measurement signal is generatedbased on the formation of the differential quotient from the firstoptical measurement signal absorbance A1 and the second opticalmeasurement signal absorbance A2 and the third optical measurementsignal absorbance A3 ((A1−A3)/(A2−A3)) in the wavelength range of390-420 nm (A1), in the wavelength range of 530 to 560 nm (A2), and inthe wavelength range of 620-890 nm (A3).
 24. The method according toclaim 19, wherein fluorescent dyes are used as dyes for the indicators.25. The method according to claim 24, wherein methylumbelliferone, asfirst indicator, is added only to the sample liquid or reagent liquid,and wherein fluorescein, as second indicator, is added in an identicalconcentration to the sample liquid or reagent liquid and to the diluent.26. The method according to claim 24, wherein the normalized opticalmeasurement signal is generated based on the formation of the quotient(Flu1/Flu2) from the first optical measurement signal (fluorescenceFlu1) and the second optical measurement signal (fluorescence Flu2) fornormalizing the fluorescence intensities at 440-470 nm (excitation at340-370 nm, Flu1) and the fluorescence intensities at 520-550 nm(excitation at 470-500 nm, Flu2).
 27. The method according to claim 1,wherein the intensity deviations of the normalized first opticalmeasurement signals of every well of the microplate from the opticalmean intensity are presented as a false-color presentation in a matrixcorresponding to the channel geometry of the liquid handling technologyfor purposes of a clear visual evaluation.
 28. The method according toclaim 1, wherein and the pipetting volumes of every well of themicroplate which are determined for determining the volume accuracy forcharacterizing the liquid handling technology are presented in a matrixcorresponding to the channel geometry of the liquid handling technology.29. The method according to claim 1, wherein with respect to type,volume and added reagents of sample liquid or reagent liquid anddiluent, with respect to the dispensing of samples into dry wells orinto liquids, and with respect to the handling of samples in themicroplates themselves, conditions are realized which come as close aspossible to a prescribed use of the liquid handling technology to becharacterized and of the microplate.